Saponin adjuvant compositions and methods relating thereto

ABSTRACT

An adjuvant composition which comprises an anionic macromolecule component particularly an ionic polysaccharide such as DEAE-dextran, and a saponin component, particularly an immunostimulating complex component. Immunogenic compositions comprising an immunogen and this adjuvant composition are also disclosed together with methods of use thereof.

FIELD OF THE INVENTION

The present invention relates generally to an adjuvant compositionhaving high adjuvant properties and low reactogenicity. The adjuvantcomposition of the present invention is useful where the development ofreactogenicity following administration of an immunogenic composition isunacceptable, for example, but not limited to, administration of an LHRHcomposition as a prophylactic and/or therapeutic agent for themodification of fertility and behaviour patterns of domestic pets orlivestock destined for consumption.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

Adjuvants are included or added to vaccines and other immunogenicformulations, to increase and in some cases direct the immune response(see reviews by Gupta, 1998, Cox and Coulter, 1992, Azuma, 1992 andAguado et al, 1999). It will be appreciated that some adjuvants, forexample the oil in water and water in oil emulsions, are widelyconsidered as strong adjuvants as they stimulate high levels ofantibody, but the reactogenicity of such adjuvants precludes theirroutine use in many animal species or in man. The unacceptablereactogenicity has been demonstrated in many animals with thetraditional Freunds' oil in water emulsion, to the extent that the useof this adjuvant in animals has been prohibited or discouraged in somecountries, even for experimental purposes. The use of a combined oil inwater/DEAE-dextran adjuvant in cattle (Hodgkinson et al, 1990) resultedin significant and undesirable reactions in 30% of the vaccinatedanimals.

The most widely used adjuvants in man and animals are based on insolublealuminium salts, particularly the hydroxide and phosphate forms,commonly and collectively termed “alum”. The recent review by Aguado etal (1999) highlights the ongoing need for stronger adjuvants in both manand animals for the development of more effective vaccines.

For example, with a wide range of soluble antigens, the immune responseto such an antigen delivered alone is very poor, and in many cases isundetectable even after two vaccinations. Such antigens require the useof an adjuvant to stimulate a consistent immune response. It will beappreciated that known adjuvants offer a range of abilities to stimulatean immune response, most usually defined in terms of antibody responseto the immunising antigen.

For haptens coupled to a carrier protein, it is widely recognised that apowerful adjuvant is required. For example, the hypothalmic peptidehormone LHRH is a 10 amino acid peptide that by itself isnon-immunogenic. Coupling of LHRH to carrier proteins, for examplediphtheria toxoid or ovalbumin provides the necessary T-cell help for animmune response to the LHRH hapten. For other large molecular weight andcomplex antigens, the T cell help is usually provided by T-cell epitopeswithin the molecule itself.

The use of DEAE-dextran as an adjuvant has been well described in thescientific literature. Wittman et al (1975), used DEAE-dextran toadjuvant a Foot and Mouth Virus (FMDV) vaccine in pigs, and showed thatit induced very high levels of immunity. Houston et al (1976) used thisadjuvant at a dose rate of 1-5 mg/kg body weight, to vaccinate monkeyswith an equine virus and showed that it induced high titres of antibody.Beh and Lascelles (1985) have also used this adjuvant to immunise sheepwith ovalbumin, and showed that it induced higher antibody titres thanother soluble adjuvants. None of these studies considered the sitereactivity of DEAE-dextran, but they did describe the significantadjuvant effects of DEAE-dextran in these animal species. DEAE-dextranhas been used in deer with keyhole limpet haemocyanin (KLH) antigen(Hibma and Griffin, 1992). In this study, DEAE-dextran induced greaterIgG responses to KLH than did Freunds adjuvant or alum.

The adjuvant properties of saponin have been long known, as has itsability to increase antibody titres to immunogens. As used herein, theterm “saponin” refers to a group of surface-active glycosides of plantorigin composed of a hydrophilic region (usually several sugar chains)in association with a hydrophobic region of either steroid ortriterpenoid structure. Although saponin is available from a number ofdiverse sources, saponins with useful adjuvant activity have beenderived from the South American tree Quillaja saponaria (Molina).Saponin from this source was used to isolate a “homogeneous” fractiondenoted “Quil A” (Dalsgaard, 1974).

Dose-site reactivity is a major concern for both the veterinary andhuman use of Quil A in vaccine preparations. One way to avoid thistoxicity of Quil A is the use of immunostimulating complexes (known asIscoms™, an abbreviation for Immuno Stimulating COMplexes). This isprimarily because Quil A is less reactive when incorporated intoimmunostimulating complexes, because its association with cholesterol inthe complex reduces its ability to bind to cholesterol in cell membranesand hence its cell lytic effects. In addition, a lesser amount of Quil Ais required to generate a similar level of adjuvant effect.

The immunomodulatory properties of the Quil A saponins and theadditional benefits to be derived from these saponins when they areincorporated into an immunostimulating complex have been described invarious publications, e.g. Cox and Coulter, 1992; Dalsgaard, 1974;Morein et al., Australian Patent Specifications Nos. 558258, 589915,590904 and 632067.

Vaccination against the hypothalamic hormone luteinising hormonereleasing hormone (referred to herein as “LHRH”, also known as GnRH) hasbeen demonstrated as an immunological method of controlling reproductionsince the early 1970's (Fraser 1975, Jeffcoate et al 1982). Eliciting animmune response to LHRH prevents the release from the anterior pituitaryof the hormones LH and FSH, which are required for the development andmaintenance of the gonads—the testes in the male and ovaries in thefemale. Thus reduction of LH and FSH levels leads to loss ofreproductive function.

De-sexing (or neutering) operations are the most widely practisedsurgical procedures in veterinary medicine and livestock animalmanagement. A significant proportion of both sexes of domestic livestockand companion animals are routinely surgically de-sexed to prevent avariety of undesirable characteristics which accompany sexual maturity.The traits include fighting, wandering, sexual behaviour, loss ofcondition, tumours of reproductive organs and pregnancy.

The control of mating behaviour by vaccination with LHRH-conjugatevaccines in companion animals such as dogs, cats and horses, and inlivestock specifically in male pigs and male and female cattle, has beenidentified as a goal as significant as the control of fertility.

Similarly, the control and treatment of disorders of the gonads andother reproductive organs, of both humans and animals, such astesticular cancer, breast cancer, prostate cancer, ovarian cancer,prostate enlargement or endometriosis is of significance.

International Patent Application No. PCT/AU98/00532, the contents ofwhich are incorporated herein by reference, discloses that the efficacyof vaccination against LHRH is significantly improved when LHRH isadministered as a conjugate with diphtheria toxoid and an ionicpolysaccharide such as DEAE-dextran. However, although the efficacy isimproved, this formulation nevertheless induces reactogenicity such asvisible swelling, that may be long-lasting, at the site ofadministration. In some instances, such as where the formulation is usedto vaccinate domestic pets (for example, dogs) or livestock destined forconsumption the occurrence of such reactogenicity following vaccinationagainst LHRH utilising this formulation is unacceptable. Accordingly,there is a need to develop a LHRH vaccine which exhibits both efficacyand low reactogenicity.

In one aspect of the work leading up to the present invention, it hasbeen determined that reactogenicity of LHRH conjugated to diphtheriatoxoid and an ionic polysaccharide can be reduced when a proportion ofthe ionic polysaccharide component is replaced with a saponin component,particularly an immunostimulating complex.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

Sequence Identity Numbers (SEQ ID NOS.) for the nucleotide and aminoacid sequences referred to in the specification are defined followingthe bibliography. A summary of the SEQ ID NOS. is provided after theExamples.

One aspect of the present invention relates to an adjuvant compositionwhich comprises an ionic macromolecule component and a saponincomponent, particularly an immunostimulating complex component.

Such an adjuvant composition has been found to have high adjuvantactivity combined with low reactogenicity. The advantage of thiscombined adjuvant over other known adjuvants is that it possesses anunusual combination of properties including:

a) an ability to elicit a very strong immune response to a range ofantigens andb) to have at the same time very low reactogenicity or reactivity,c) the reactogenicity is lower than that of the more reactogeniccomponent in the combination, andd) the adjuvant effect or immunostimulating effect is greater than thatof either of the component adjuvants alone.

In yet another aspect there is provided an adjuvant composition whichcomprises an ionic polysaccharide component and a saponin component,particularly an immuno-stimulating complex component.

In still yet another aspect there is provided an adjuvant compositionwhich comprises an ionic polysaccharide component and a saponincomponent, particularly a protein-free immunostimulating complexcomponent.

In a further aspect of the present invention there is provided animmunogenic composition comprising an immunogen and an adjuvantcomposition, which adjuvant composition comprises an ionic macromoleculecomponent and a saponin component, particularly an immunostimulatingcomplex component.

In another further aspect there is provided an immunogenic compositioncomprising an immunogen and an adjuvant composition, which adjuvantcomposition comprises an ionic polysaccharide component and a saponincomponent, particularly an immunostimulating complex component.

In still another further aspect there is provided an immunogeniccomposition comprising an immunogen and an adjuvant composition, whichadjuvant composition comprises an ionic polysaccharide component and asaponin component, particularly a protein-free immunostimulating complexcomponent.

In still another further aspect of the present invention there isprovided a pharmaceutical composition comprising an immunogeniccomposition as broadly described above, together with one or morepharmaceutically acceptable carriers and/or diluents.

Another aspect of the present invention relates to a method ofeliciting, in an animal, an effective immune response, said methodcomprising administering to said animal an effective amount of animmunogenic composition as broadly described above.

In yet another aspect, the present invention provides the use of anadjuvant composition as broadly described above in the manufacture of acomposition for eliciting an effective immune response in an animal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the determination thatwhen the ionic polysaccharide component of the vaccine comprising LHRHconjugated to diphtheria toxoid as disclosed in International PatentApplication No. PCT/AU98/00532 is combined with an immunostimulatingcomplex, the reactogenicity of the immunogenic composition is reducedwithout significant reduction in efficacy.

Accordingly, one preferred aspect of the present invention relates to animmunogenic composition for use in eliciting an effective immuneresponse to LHRH, said composition comprising a LHRH-diphtheria toxoidconjugate and an adjuvant composition, which adjuvant compositioncomprises an ionic polysaccharide or other ionic macromolecule componentand a saponin component, particularly an immunostimulating complexcomponent.

The development of reactogenicity to an immunogenic preparation, such asat the site of administration of a vaccine, is usually assessed byreference to the development of a number of symptoms including symptomsof inflammation such as swelling which is detectable either by palpationor, if more severe, by the eye, redness, and abscess formation. Thedegree of reactogenicity is usually determined by reference to theoccurrence, duration and severity of any one or more of these symptoms.For example, reactogenicity which results in visible abscess formationis of greater severity than reactogenicity which involves only swelling.Further, swelling which is visible to the eye is a more severe form ofreactogenicity than swelling which is detectable only by palpation. Inaccordance with the present invention, reference to reactogenicity whichis “low” should be understood as reactogenicity which produces either nodetectable symptoms or symptoms which are not visible to the eye. Forexample, swelling which is detectable only by palpation is an example ofreactogenicity which is low. The present invention should be understoodto extend to the complete absence of any reactogenicity. In the contextof the present invention, low reactogenicity may also be taken toinclude visible swelling of only short duration.

Reference to an “ionic macromolecule” or an “ionic polysaccharide”should be understood as a reference to any positively or negativelycharged polysaccharide or other macromolecule, or derivative or chemicalequivalent thereof. Reference to “derivative” and “chemical equivalent”should be understood to have the same meaning as outlined below.

The polysaccharide or other macromolecule may be chemically modified tointroduce charged groups, so that it becomes highly charged. In solidform or when in solution, the charged groups are paired with a counterion so that the overall net charge is zero or close to zero. Thepolysaccharide may be selected from any one of a number of suchmolecules, preferably soluble molecules to aid in sterilisation forexample by filtration, but the polysaccharide is not restricted tosoluble polysaccharides, as insoluble materials may be sterilised byirradiation, wet or dry heat or other processes that do not rely onsolubility. Preferably the polysaccharide is a polymer of glucose,linked alpha 1-6 to form a soluble dextran, although insoluble forms ofdextran linked alpha 1-3 may also be used.

The polysacharide or other macromolecule may be modified to give strongpositive (+ve) charged groups at pH values between 5.0 and 8.0. Themodifying group can be selected from any one of a number of +ve chargedgroups, but preferably is a diethyl-aminoethyl (DEAE) group. Analternative +ve charged polysaccharide is QAE-dextran. The modifieddextran known as DEAE-dextran, may have a molecular size in the range50,000 Da to 5×10⁶ Da, preferably a molecular weight range of 500,000 to1.5×10⁶ da. It is known that such large macromolecules as dextran areheterogeneous in size, typically with 95% of the material withinapproximately a 4 fold size range. The level of modification is high,such that on average every third glucose residue is substituted with aDEAE group. Such material may be purchased commercially or manufacturedfrom dextran. Alternatively, the dextran or other polysaccharide may benegatively charged, for example by modification with sulphate groups.Preferably, the ionic polysaccharide component of the adjuvantcomposition of the present invention is a DEAE-dextran component.

The saponin component may, for example, be Quil A or another purified orsemi-purified saponin preparation (including a mixture of saponinfractions). Preferably, the saponin component is provided in the form ofan immunostimulating complex.

lmmunostimulating complexes (or Iscom™) which are incorporated in theadjuvant composition in accordance with the present invention may beprepared by techniques which are well known to persons skilled in theart, and which are described in detail in the publications of Cox andCoulter, 1992 and Morein et al., Australian Patent Specifications No.558258, 589915, 590904 and 632067 the disclosures of which areincorporated herein by reference.

Briefly, immunostimulating complexes are typically, but not limited to,small cage like structures 30-40 nM in diameter. The final formulationof an immunostimulating complex with an optimal amount of protein is amolar ratio of Quil A, cholesterol, phosphatidyl choline and protein ina ratio of 5:1:1:1. An immunostimulating complex may contain, forexample, 5 to 10% by weight Quil A, 1 to 5% cholesterol andphospholipids and the remainder protein. Peptides can be incorporatedinto the immunostimulating complex either directly or by chemicalcoupling to a carrier protein (e.g. diphtheria toxiod or influenzaenvelope protein) after incorporation of protein into immunostimulatingcomplexes. Reference to an “immunostimulating complex” should beunderstood to include reference to derivatives, chemical equivalents andanalogues thereof. For example, reference to a derivative of animmunostimulating complex includes reference to an immunostimulatingcomplex in which one or more of Quil A, cholesterol, phosphatidylcholine or protein, for example, are deleted or where a component inaddition to Quil A, cholesterol, phosphatidyl choline or protein isadded to the complex. The functional equivalent of an immunostimulatingcomplex may be an immunostimulating complex in which one or more of itsfour components are replaced with a functional equivalent. In apreferred embodiment of the present invention, the protein component ofthe immunostimulating complex is deleted. This type of immunostimulatingcomplex is herein referred to as a “protein-free immunostimulatingcomplex” (or Iscomatrix™).

The immunostimulating complex in the adjuvant composition of the presentinvention should be oppositely charged to the ionic polysaccharidecomponent. Thus in the preferred combination with positively chargedDEAE-dextran, the immunostimulating complex is negatively charged. Thisdesirable characteristic may be achieved by incorporation of chargedsaponins into the structure. Formation of an effective adjuvantcomposition by combining an immunostimulating complex with negativelycharged polysaccharide component would require that theimmunostimulating complex be positively charged. In an alternate form, apositively charged immunostimulating complex would be combined with anegatively charged ionic-polysaccharide component.

A wide range of antigens, or other immunogens may be combined with thenovel adjuvant composition of the present invention, including solubleprotein antigens, peptide hapten conjugated to a carrier protein andwhole virus as antigen. In addition, prior knowledge of adjuvants andtheir activity, other antigens such as recombinant proteins, antigenicpolysaccharides, antigenic lipids, peptides and other particulateantigens may also be suitably used with this adjuvant composition.

The final formulation is to be used to raise an immune response againstthe antigen of choice. Both immunostimulating complexes loaded withantigen or protein-free immunostimulating complexes may be used to formthe adjuvant composition. The combination adjuvant may be formed withantigen first incorporated into the immunostimulating complex. In analternate form, antigen may be added to the combination adjuvant afterformulation of the adjuvant. This has many advantages in manufacture ofvaccines, both experimentally and commercially, as the one form ofadjuvant may be used for a wide range of antigens.

DEAE-dextran is known in the art to be a very strong adjuvant, but oftenwith unacceptable reactogenicity. Immunostimulating complexes are knownto be acceptable adjuvants on their own, but with a far lesser degree ofimmunostimulating activity when compared to DEAE-dextran. Morespecifically, the new adjuvant is a carefully controlled combination ofDEAE-dextran and immunostimulating complex, the controlled ratio beingthat which maintains the adjuvant activity of both components,particularly of the more powerful adjuvant DEAE-dextran but also ofimmunostimulating complexes, but the combination is such that theunwanted reactogencicity of the DEAE-dextran is neutralised. In the art,it is expected that combinations of adjuvants will be more reactogenicthan the individual components. Such is the case with DEAE-dextrancombined with oil emulsions (Hodgkinson et al, 1990).

Extensive experimentation in a range of animal species has shown thatthe mass ratio of ionic polysaccharide such as DEAE-dextran over that ofimmunostimulating complex may be critical to obtaining a combinationadjuvant of low reactogenicity. Reducing the level of DEAE-dextran by 10fold would result in a combination adjuvant of weak activity, but alsolow reactogenicity. As demonstrated in the Examples, using a higherlevel of DEAE-dextran with a lower ratio of immunostimulating complexes,e.g. a mass ratio of 208, results in a combination adjuvant of slightlylower immunostimulating activity, but unacceptably higherreactogenicity.

The mass ratios referred to herein relate to the preferred adjuvantcomposition comprising DEAE-dextran in which the dextran is substitutedabout every third glucose residue, and a protein-free immunostimulatingcomplex having a Quil A:cholesterol: phospholipid ratio of 5:1:1.

Formulations with a mass ratio in the range of 50 to 300 have been foundto be effective, with a preferred range of 100 to 140. A preferredformulation is one with a mass ratio of DEAE-dextran toimmunostimulating complex of 125. This is exemplified by vaccinescontaining about 10 mg DEAE-dextran and about 80 μg immunostimulatingcomplex for use in smaller or more sensitive animals such as dogs orman, or similarly about 100 mg DEAE-dextran with about 800 μgimmunostimulating complex, or about 150 mg DEAE-dextran with about 500μg immunostimulating complex, for use in larger animals such as horses,cattle, sheep and pigs.

Reference to an “effective” immune response should be understood as areference to an immune response which either directly or indirectlyleads to a desired prophylactic or therapeutic effect. In the case wherethe immunogen comprises a LHRH-diphtheria toxin conjugate, such aresponse includes the reduction or complete blocking of reproductivefunction (i.e. reduces or prevents the development of or functioning ofany one or more of the reproductive organ's activities or modulates thehormonal levels of an animal such that any one or more aspects ofreproduction or reproductive activity are reduced) in at least 90%, andpreferably at least 95%, of animals treated. It should be understoodthat efficacy is a functional measure and is not defined by reference toanti-LHRH antibody titre alone since the presence of circulatingantibody alone is not necessarily indicative of the capacity of saidcirculating antibody to block reproductive function. The term“reproductive organ” should be understood in its broadest sense to referto the male and female gonads and accessory sexual organs. “Accessorysexual organs” should also be understood in its broadest sense andincludes, for example, the prostate, breasts, seminal vesicles and theuterus.

Reference hereinafter to “LHRH” should be read as including reference toall forms of LHRH and derivatives, equivalents and analogues thereof.

Reference to “derivatives, equivalents and analogues” should beunderstood to include reference to fragments, parts, portions, chemicalequivalents, mutants, homologues and analogues from natural, syntheticor recombinant sources, including fusion proteins. For example, withrespect to LHRH, said LHRH includes peptides comprising a sequence ofamino acids substantially as set forth in SEQ ID NO:1 or SEQ ID NO:2 orSEQ ID NO:3 or SEQ ID NO:4 or having at least 50% similarity thereto.The molecules defined in SEQ ID Nos:1, 2 and 3 are from the human andare conserved across all mammals. SEQ ID NO:4 is a derivative of SEQ IDNO:2 wherein spacers have been introduced at the N-terminus. Chemicalequivalents of LHRH can act as a functional analogue of LHRH. Chemicalequivalents may not necessarily be derived from LHRH but may sharecertain similarities. Alternatively, chemical equivalents may bespecifically designed to mimic certain physiochemical properties ofLHRH. Chemical equivalents may be chemically synthesised or may bedetected following, for example, natural product screening.

Homologues of LHRH contemplated herein include, but are not limited to,LHRH derived from different phyla including birds, fish, reptiles andinvertebrates.

“Derivatives” may also be derived from insertion, deletion orsubstitution of amino acids. Amino acid insertional derivatives includeamino and/or carboxylic terminal fusions as well as intrasequenceinsertions of single or multiple amino acids. Insertional amino acidssequence variants are those in which one or more amino acid ornon-natural amino acid residues are introduced into a predetermined sitein the protein although random insertion is also possible with suitablescreening of the resulting product. Deletional variants arecharacterised by the removal of one or more amino acids from sequence.Substitutional amino acid variants are those in which at least oneresidue in the sequence has been removed and a different natural ornon-natural residue inserted in its place. Typical substitutions arethose made in accordance with Table 1:

TABLE 1 Suitable residues for amino acid substitutions Original ResidueExemplary Substitutions Ala Ser * Arg Lys Asn Gln; His Asp Glu Cys SerGln Asn * Glu Ala * Gly Pro * His Asn; Gln Ile Leu; Val * Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr * Ser Thr Thr Ser * TrpTyr * Tyr Trp; Phe Val Ile; Leu Key: Amino acid residues marked with anasterisk indicate residues present in the mammalian LHRH sequence.

Examples of non-natural amino acids include, but are not limited to theD-isomers of said amino acids. “Additions” to amino acid sequencesinclude fusion with other peptides, polypeptides or proteins.

Reference to diphtheria toxoid should be understood as a reference toall forms of diphtheria toxoid and derivatives thereof. The term“derivatives” has the same meaning as hereinbefore defined. Derivativesmay include, for example, molecules comprising the diphtheria toxoid Tcell epitopes or said T cell epitopes in isolation.

Preferably, said LHRH comprises an LHRH C-terminal fragment of at leastfive amino acids. More preferably, said LHRH is full length LHRH or“LHRH 1-10 form” which comprises the amino acid sequence substantiallyas set forth in SEQ ID NO:1. Even more preferably, said LHRH comprisesthe amino acid sequence substantially as set forth in SEQ ID NO:2 andwherein the carboxyl terminus of said amino acid sequence is amidated.Said preferred LHRH is referred to herein as “LHRH 2-10 form”.

Accordingly, in one preferred embodiment, there is provided acomposition for use in eliciting an effective immune response to LHRHcomprising a LHRH 2-10 form-diphtheria toxoid conjugate and an adjuvantcomposition, which adjuvant composition comprises an ionicpolysaccharide component (preferably a DEAE-dextran component) and asaponin component, particularly an immunostimulating complex component.

In another embodiment, there is provided a composition for use ineliciting an effective immune response to LHRH comprising a LHRH 2-10form-diphtheria toxoid conjugate and to an adjuvant composition, whichadjuvant composition comprises an ionic polysaccharide component(preferably a DEAE-dextran component) and a saponin component,particularly a protein-free immunostimulating complex component.

Although not intending to limit this embodiment of the invention to anyone method, the LHRH peptide may be synthesised by Fmoc chemistry andthe resulting peptide coupled to the carrier protein diphtheria toxoid.Said coupling may be performed as described in Ladd et al 1990 or inBonneau et al 1994, and the resulting conjugate of peptide and carrierprotein (referred to herein as “peptide-conjugate”) processed to be freeof unbound peptide and other by-products of conjugation. Such processingmay be achieved by conventional dialysis or by ultrafiltration. Theresulting peptide-conjugate is adsorbed to the ionic polysaccharideadjuvant.

Still without limiting the present invention to any one theory or modeof action, administration of an effective amount of the LHRH preparationof the present invention induces a significantly more effective immuneresponse against LHRH than the LHRH-carrier-adjuvant compositionsdescribed in the prior art. Said improved efficacy is observed when theimmunogenic LHRH composition specifically comprises the carrierdiphtheria-toxoid and an ionic polysaccharide adjuvant. By replacing aportion of the ionic polysaccharide component with an immunostimulatingcomplex component, the reactogenicity of the LHRH-conjugate preparationcan be reduced while maintaining its efficacy.

LHRH-conjugate preparations suitable for use in accordance with thepresent invention preferably comprise 5-500 μg of LHRH-diphtheria toxoidconjugate in 5-500 mg ionic polysaccharide (such asDEAE-dextran)/40-4000 μg immunostimulating complex (such asIscomatrix™).

Pharmaceutical compositions in accordance with the present invnetion maybe administered by injection by any of the routes currently known in theart of vaccinology, e.g. by subcutaneous, intramuscular, intradermalinjection or by oral administration, or by application to mucosalmembranes, either by direct application in solution or gel or othersuitable formulation, or by aerosol administration to the nasal orrespiratory surfaces. Preferably the formulation is administered bysubcutaneous or intramuscular injection.

The formulation may be stored as a solid, frozen or dried, either byfreeze drying or lyophilisation, or by spray drying or other forms ofdrying.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or salts such as sodium chloride. Prolonged absorption or delayedrelease of the injectable compositions can be brought about by the usein the compositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying, the freeze-dryingtechnique and the spray-drying technique which yield a powder of theactive ingredients plus any additional desired ingredient frompreviously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or they may be enclosed in hard or soft shell gelatincapsule, or compressed into tablets, or incorporated directly with thefood of the diet. For oral administration, the active compound may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. The percentage of the compositions and preparations may,of course, be varied and may conveniently be between about 5 to about80% of the weight of the unit. The amount of active compound in suchuseful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter: a binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; and a lubricant such as magnesium stearate. When thedosage unit form is a capsule, it may contain, in addition to materialsof the above type, a liquid carrier. Various other materials may bepresent as coatings or to otherwise modify the physical form of thedosage unit. A syrup or elixir may contain the active compound, methyland propylparabens as preservatives, and a dye. Of course, any materialused in preparing any dosage unit form should be veterinarily pure andsubstantially non-toxic in the amounts employed. In addition, the activecompound(s) may be incorporated into sustained-release preparations andformulations.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for veterinarily active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.For administration to livestock it is particularly advantageous to use amulti-dose container linked to a repeating vaccination gun. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subjects to be treated; each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the novel dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from 0.5 μg to about 2000 μg.Expressed in proportions, the active compound is generally present infrom about 0.5 μg to about 2000 μg/ml of carrier. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

Reference to “animal” should be understood as the reference to allanimals including primates (e.g. humans, monkeys), livestock animals(e.g. sheep, cows, horses, donkeys, goats, pigs), laboratory testanimals (e.g. rats, guinea pigs, rabbits, hamsters), companion animals(e.g. dogs, cats), captive wild animals (e.g. emus, kangaroos, deer,foxes), ayes (e.g. chickens, ducks, bantams, pheasants, emus,ostriches), reptiles (e.g. lizards, snakes, frogs) and fish (e.g. trout,salmon, tuna). Said animal may be male or female.

Further features of the present invention are more fully described inthe following Examples. It is to be understood, however, that thisdetailed description is included solely for the purposes of exemplifyingthe present invention. It should not be understood in any way as arestriction on the broad description of the invention as set out above.

Example 1 Preparation of LHRH-Conjugate Preparation

The LHRH-conjugate is based on a synthetic 2-10 form of LuteinisingHormone Releasing Hormone (LHRH) peptide coupled to a carrier protein.The peptide by itself is too small to be antigenic, and coupling to acarrier protein is required so that the peptide acts as a hapten andimmunity is induced to LHRH. The carrier protein is diphtheria-toxoid.

The peptide is synthesised by Fmoc chemistry and the resulting 2-10 formLHRH peptide is coupled to diphtheria toxoid. The coupling may beperformed as described in Ladd et al. 1990 or in Bonneau et al. 1994,and the resulting conjugate of peptide and diphtheria-toxoid processedto be free of unbound peptide and other by-products of conjugation. Suchprocessing may be achieved by conventional dialysis or byultrafiltration.

The resulting LHRH-carrier protein preparation may be used to prepare acomposition for administration by formulation with or in an adjuvant.The levels of active component and adjuvant are in part dependent on thespecies being targeted and are chosen to achieve the desired level andduration of the immune response required and on the lack ofreactogenicity (ie tissue compatibility).

Example 2 A. Preparation of Protein-Free Immunostimulating Complex(Iscomatrix™)

Quil A (Superfos, Denmark) solution (sterile) and dipalmitoylphosphatidyl choline (DPPC)/cholesterol solution in 20% Mega 10detergent are aseptically combined under GMP conditions in a steriletemperature controlled reaction vessel in quantities calculated toresult in starting proportions of 5:1:1 for Quil A:DPPC:cholesterol (byweight). After reaction, the preparation is ultrafiltered to removeunincorporated starting materials and free Mega 10 detergent. Thispreparation, known as Iscomatrix™, is essentially a preformed adjuvantconsisting of protein-free immunostimulating complex particles.

B. Preparation of Combination Adjuvant Comprising DEAE-Dextran andIscomatrix™

DEAE-dextran (Pharmacia, MW range 500,000 to 1,500,000) is simplyprepared as a 20% w/v solution by first dissolution in water, the pHadjusted to 7.5 with NaOH, the volume adjusted to give the requiredconcentration and then sterilised by filtration.

Iscomatrix™ and DEAE-dextran plus antigen are then mixed in theappropriate ratios, followed by the addition of diluent to adjust thefinal volume to achieve the correct concentration of all of thecomponents of the vaccine.

An appropriate volume of DEAE-dextran is first added to a sterile mixingvessel containing a suitable stirring device. For volumes less than 2litres, a magnetic stirring bar is suitable. The appropriate volume ofIscomatrix™ (˜2 mg/mL) is then added, followed by the appropriatevolumes of antigen and then diluent to bring the material to the correctvolume. The diluent of choice is of low ionic strength, to bring thewhole final formulation to approximate isotonicity and thus avoid painon injection due to hyper-tonicity or higher than physiologicalosmolarity. This is of particular importance in the art of vaccineformulation, as severe pain resulting from injection is highlyundesirable and would render the whole formulation unsuitable. Thediluent found to be suitable for a canine vaccine is sterile water forinjection, ie pyrogen free distilled water.

Example 3 A. Preparation of Vaccines for Dog Trials

a. DEAE-Dextran Adjuvant Alone.

The formulation required to be tested in this trial was a 1 mL dosecontaining 50 mg DEAE-dextran, 200 μg LHRH-diphtheria toxoid conjugate,with thiomersal (0.01%) added as preservative, in sterile phosphatebuffered saline.

The adjuvant for this vaccine is prepared by dissolving DEAE-dextran indistilled water and adjusting the pH to 7.5 with sodium hydroxide. Thefinal concentration of the DEAE-dextran is adjusted to 20% w/v by theaddition of distilled water, after pH adjustment. The solution is thensterile filtered. LHRH peptide (2-10 LHRH) is conjugated to diphtheriatoxoid as described in Example 1. The conjugate preparation is sterilefiltered and the protein concentration determined by standard methods,e.g. the BCA protein assay. The preparation used for this study was 5.7mg protein/mL.

b. Iscomatrix™ Adjuvant Alone.

The formulation required to be tested in this trial was a 1 mL dosecontaining 60 μg Iscomatrix™, 200 μg LHRH-diphtheria toxoid conjugate,with thiomersal (0.01%) added as preservative in sterile phosphatebuffered saline.

The adjuvant for this vaccine, Iscomatrix™, is prepared as described inExample 2A using Quil A as the saponin derivative, together withcholesterol, dipalmitoyl phosphatidyl choline (DPPC) and Mega 10 as thedetergent. The process is performed under sterile conditions with allcomponents sterilised prior to addition. The Quil A concentration wasdetermined to be 2.24 mg/ml.

LHRH peptide (2-10 LHRH) is conjugated to diphtheria toxoid as describedin Example 1. The conjugate preparation is sterile filtered and theprotein concentration determined by standard methods, e.g. the BCAprotein assay. The preparation used for this study was 5.7 mgprotein/mL.

c. Iscomatrix™ DEAE-Dextran

The formulation required to be tested in this trial was a 1 mL dosecontaining 60 μg Iscomatrix™+10 mg DEAE-dextran, 200 μg LHRH-diphtheriatoxoid conjugate, with thiomersal (0.01%) added as preservative, insterile phosphate buffered saline.

The adjuvant for this vaccine is a compound adjuvant, obtained bycombining appropriate proportions of Iscomatrix™ and DEAE-dextran.Iscomatrix™, is prepared as described in Example 2A. DEAE-dextran isprepared as described in above.

LHRH peptide (2-10 LHRH) is conjugated to diphtheria toxoid as describedin Example 1. The conjugate preparation is sterile filtered and theprotein concentration determined by standard methods, e.g. the BCAprotein assay. The preparation used for this study was 4.9 mgprotein/mL.

B. Vaccination of Dogs

All trials were conducted in an identical manner.

Dogs of 6-12 months of age and of mixed sex were housed in indoor penswith free access to outdoor runs. They were fed a commercially availablebalanced diet.

Dogs (groups of 7) were vaccinated subcutaneously in the scruff of theneck at 0 and 4 weeks with a 1 mL dose of vaccine. All vaccines wereadministered from a 2 mL syringe fitted with a 23 gauge needle.

Dogs were bled at regular intervals to monitor antibody levels to LHRH.Reactogenicity of the vaccines was determined by close examination ofthe dogs after vaccination.

Examination was by visual inspection and by thorough manual palpation ofthe vaccine site and surrounding area.

Site reactions were scored by close visual examination and physicalpalpation of the injection site and surrounding area. Reactions werescored as:

-   -   0 No visible or detectable reaction    -   1 Reaction only detectable by palpation    -   2 Visible reaction detectable without palpation    -   3 Severe abscessed reaction

Site Reaction Scores

Vaccine 1 Week PB 2 Weeks PB 4 Weeks PB Formulation 0 1 2 0 1 2 0 1 2DEAE-dextran (50 mg) 3/7 2/7 2/7 3/7 4/7 — 7/7 — — Iscomatrix ™ (60 μg)7/7 — — 7/7 — — 7/7 — — DEAE-dextran (10 mg) + 5/7 1/7 1/7 7/7 — — 7/7 —— Iscomatrix ™ (60 μg) (Note: PB = post-boost)

These results show that the DEAE-dextran formulation is much morereactive than either the Iscomatrix™ formulation or theIscomatrix™+DEAE-dextran formulation. With DEAE-dextran alone,significant reactions (Score 1 or greater) persisted for at least 2weeks. With the Iscomatrix™ formulation, reactions were not detected andwith the DEAE-dextran+Iscomatrix™ formulation, minor reactions in 2/7dogs persisted for only 1 week.

In order to provide a more quantitative indication of reactogenicity,the approximate volumes of the site reactions, when detected, weremeasured as width×length×height, in cm. The results for 1 and 2 weekpost-boost (PB) from the above table are given below. Site reactionvolumes are not given for the Iscomatrix™ vaccine formulation group, asno site reactions were detected.

Site Reaction Volumes

Week 1 PB Week 2 PB Site Reaction Site Reaction Volumes (cm³) Volumes(cm³) Vaccine Score Score Formulation 0 1 2 0 1 2 DEAE-dextran (50 mg) —32, 40, — 6.25, 1 — 4 8 0.5, 0.5 Iscomatrix ™ (60 μg) + 1.1 6 — — —DEAE-dextran (10 mg)

These results show that the degree of reactogenicity is much less in theIscomatrix™+DEAE-dextran formulation than with the DEAE-dextranformulation alone as shown by the size of the reactions.

Example 4 Vaccination of Dogs with Varying Ratios of DEAE-Dextran andIscomatrix

Beagle foxhound cross dogs (groups of 8 or 13) were vaccinated withformulations of 2-10 LHRH linked to DT. The active component wasformulated in either DEAE-dextran alone, Iscomatrix alone or in thecombination adjuvant DEAE-dextran plus Iscomatrix.

-   A) DEAE-dextran vaccines: Each dose was contained in a volume of 1    mL formulated to contain either 10 mg or 50 mg of DEAE-dextran with    200 μg 2-10 LHRH-DT conjugate.-   B) Iscomatrix: Each dose was formulated in 1 mL with either 60 μg or    150 μg Iscomatrix with 200 μg 2-10 LHRH-DT conjugate.-   C) Combination adjuvant: Each dose was contained in a volume of 1 mL    formulated to contain DEAE-dextran and Iscomatrix in the ratios    indicated below with 200 μg 2-10 LHRH-DT conjugate.

The combination adjuvant contained either:

-   1. 10 mg DEAE-dextran with 60 μg Iscomatrix—referred to as 10/60    vaccine-   2. 12.5 mg DEAE-dextran with 60 μg Iscom_(TM)atrix—referred to as    12.5/60 vaccine-   3. 10 mg DEAE-dextran with 80 μg Iscomatrix—referred to as 10/80    vaccine

Vaccination Schedule:

These formulations were administered to dogs by subcutaneous injectionat 0 and 4 weeks. Blood samples were taken at 2 weeks after the secondvaccination and assayed for antibodies to LHRH.

Site reactions were determined by careful palpation of the injectionsite and surrounding tissue at weekly intervals. The site reaction datapresented are at post boost (PB) vaccination. Reaction to vaccinationmay in certain circumstances be increased with continuing vaccination,and thus the reactogenicity post boost may be considered as morestringent test than post primary vaccination.

Site Reaction Scores

Site reactions are scored by close inspection and palpation, with thescore on the following scale:

0 No visible or detectable reaction.1 Reaction only detectable by palpation2 Visible reaction detectable without palpation3 Severe abscessed reaction

Vaccine 1 Week PB 2 Weeks PB 4 Weeks PB Formulation 0 1 2 0 1 2 0 1 2DEAE-dextran 1/8 2/8 4/8 4/8 2/8 1/8 7/8 0/8 0/8 (10 mg) DEAE-dextran +5/8 1/8 2/8 5/8 1/8 2/8 7/8 1/8 0/8 Iscomatrix ™ 12.5/60 VaccineDEAE-dextran + 11/13  2/13  0/13 10/13  3/13  0/13 12/13  1/13  0/13Iscomatrix ™ 10/80 Vaccine

Site Reaction Volumes

Volumes are calculated from the measured size of the local reactiondetermined by palpation, and are volumes are expressed in mL. Thismeasure gives a better indication of the severity of the site reactions.

Vaccine 1 Week PB 2 Weeks PB 4 Weeks PB Formulation 0 1 2 0 1 2 0 1 2DEAE-dextran — 0.12 24, — 0.25 1.7 — — — (10 mg) 5 12, 0.75 9, 3DEAE-dextran + — — 32, 0 1.5 1.8, — 0.03 — Iscomatrix ™ 2 3 12.5/60Vaccine DEAE-dextran + — 0.25 0 0.06 — — — — Iscomatrix ™ 0.5 10/80Vaccine 2

Summary:

These data in dogs with 2-10 LHRH diphtheria toxoid conjugate show thatcombination adjuvants with varying ratios of DEAE-dextran to Iscomatrixhave varying degrees of reactogenicity. Comparison of the sitereactogenicity of the various formulations indicates that, compared todextran, the combined adjuvant formulated at a ratio of 12.5 mgDEAE-dextran to 60 μg Iscomatrix is as reactogenic if not morereactogenic than the same dose of DEAE-dextran alone. This is a massratio of 208.

In contrast, the formulation containing 10 mg DEAE-dextran to 80 μgIscomatrix has significantly less reactivity than either DEAE-dextranalone or the 12.5/60 formulation. This formulation did not give visiblereactions at any time after injection, and comparison of reactionvolumes indicates that at all times after injection, the 10/80formulation far fewer reactions and those few that do occur, with a peakof 3 of 13 dogs at 2 weeks post more acceptable and of shorter duration.This has a mass ratio of 125.

The immunostimulatory effectiveness of an adjuvant may be judged by themagnitude and duration of the antibody response elicited. The antibodytitres at 8 weeks post boost are shown below for the DEAE-dextran andcombination adjuvants.

Antibody titres to LHRH (8 Weeks Post-Boost)

Vaccine Formulation Geometric mean titre Range Iscomatrix 122443761-58097 (150 μg/dose) Iscomatrix ™ 3965 3322-7343  (60 μg/dose)DEAE-dextran 38913 23166-50377  (50 mg/dose) DEAE-dextran 69172007-24477 (10 mg/dose) DEAE-dextran + 11686 4149-35112 Iscomatrix ™10/60 Vaccine DEAE-dextran + 9670 4191-54737 Iscomatrix ™ 12.5/60Vaccine DEAE-dextran + 19456 9730-50644 Iscomatrix ™ 10/80 Vaccine

Summary:

The titres in the above table show that the 10/80 vaccine (mass ratio125) is a stronger immunostimulant than either the combination adjuvant12.5/60 vaccine (mass ratio 208) or 10 mg DEAE-dextran sole adjuvantvaccine.

Of particular note is the effectiveness of the 10/80 vaccine in raisingthe minimum titre of the lowest responder dogs. Vaccine efficacy is inmany instances reduced by the ineffective response in a small proportionof the test population and is not usually determined by a maximal andeffective response in proportion of the test population.

Both of the combined adjuvant vaccines are more effective than theDEAE-dextran alone formulation or the Iscomatrix based vaccine. Theresponse of the combination adjuvant is greater than the additive effectand thus the two components may be judged to be acting in synergy. Themass ratio of 125 is more effective in increasing the lower respondingdogs.

Example 5 Vaccination of Sheep with Bovine Viral Diarrhoea Virus (BVDV)in Combination Adjuvant 1. Growth and Inactivation of Bovine ViralDiarrhoea Virus (BVDV): Growth of Viral Antigen:

Two strains of BVDV virus were used, Trangie and Bega, isolated frominfected cattle in Australia. The growth medium used was Eagles minimumessential medium containing non essential amino acids single strengthand 3% bicarbonate, 2% HEPES and 2% adult bovine serum.

A 1×1700 cm² roller bottle was inoculated with Trangie (AJ050) at amultiplicity of infection (MOI) of 0.1. The bottle was incubated withrolling for 5 days at 37° C., then frozen. A 1×1700 cm² roller bottleinoculated with Bega (WVS p3) at a MOI of 1.0 and was incubated withrolling for 5 days at 37° C., then frozen. Roller bottles were thawedand the antigen content tested by antigen specific ELISA assays. Thecharacteristics of the preparations used in the vaccine formulation areas follows:

-   -   Trangie: E2 (surface glycoprotein) level=⅛        -   NS3 (non structural protein) level=⅛    -   Bega: E2 level=⅛        -   NS3 level=¼

Inactivation:

50 mls of each virus was dispensed into a 500 ml bottleAdd 50 mls/litre of 2.8% sodium bicarbonate=5 mlsAdd 20 mls/litre of 1M HEPES buffer=2.1 mlsAdd 1 ml/litre of 13 propriolactone (BPL)=107 μl

Contents were transferred to a new 500 ml bottle containing a magneticstirrer and stirred at 2-8° C. for 21 hours. Inactivated material wastransferred to 37° C. and stirred for another 3 hours to hydrolyse anyremaining BPL.

2. Vaccine Formulation:

Combination adjuvant: Each dose comprised 2 mls of inactivated virus and1 ml of combination adjuvant.

The bulk vaccine was formulated as:

-   -   30 mls inactivated Bega and Trangie    -   15 mls of combination adjuvant, (111 mg DEAE-dextran and 880 μg        Iscomatrix/mL, Equal to a mass ratio of 125)    -   450 μl thiomersal as preservative.    -   Iscom™ adjuvant: Each dose comprised both strains of virus and 2        mg Iscom™ as Quil A equivalent, in a total volume of 2 mL.    -   The vaccines were dispensed into a bulk plastic pillow pack        after being mixed by vortexing and inversion and stored at 2-8°        C.

Vaccination Schedule:

Both vaccines were administered as indicated below:

-   Day 0: Sheep were with either 3 mls each of combination adjuvant    vaccineor 2 mls of Iscom™ vaccine, administered subcutaneously in    the neck.-   Day 28: Each animal given a second dose of vaccine administered as    above.-   Groups: 10 sheep were vaccinated with the Iscom™ adjuvanted vaccine.    -   4 sheep were vaccinated with the combination adjuvant vaccine.

Site Reactions:

Combination adjuvant: Sheep were examined at 7 day intervals after theprimary and secondary vaccination. One of the 4 sheep developed a score1 site reaction (small swelling palpable, not visible) after the primaryvaccination. This was resolved by 2 weeks post vaccination.

Iscom™: Sheep were examined at 6 days after boost. 7 of 10 sheep hadlarge reactions at the site of injection.

Summary: The combination adjuvant had smaller and less frequent sitereactions, indicating that it induced lower levels of reactogenicity.

Example 6 Vaccination of Mice with Ovalbumin in Combination Adjuvant

C57/Blk6 mice (n=3 per grp) were vaccinated subcutaneously with chickenegg albumin (ovalbumin, OVA) formulated in either:

-   1. 0.625 mg DEAE-dextran-   2. 5 μg Iscomatrix-   3. 0.625 mg DEAE-dextran with 5 μg Iscomatrix. This is a vaccine    containing the preferred combined adjuvant of mass ratio formulation    of 125.

Each dose of the above vaccines contained 12.5 μg ovalbumin in a 100 μLvolume, the volume and concentration of the components being adjustedwith sterile water for injection.

The mice were vaccinated only once subcutaneously and blood samplestaken 14 days later after the primary vaccination at euthanasia. Sera soobtained were assayed for IgM and IgG responses to OVA.

Mice were monitored for health and signs of systemic reactogenicity.

Reactogenicity:

Mice in the DEAE-dextran and combination adjuvant groups remained wellfor the 14 days after primary vaccination.

However, 2 of the 3 mice vaccinated with Iscomatrix died with 8 days ofvaccination. Toxicity of Iscoms™, saponins and Iscomatrix to mice iswell known. The mice in the combination adjuvant group remained well,indicating that the combination adjuvant is less reactogenicsystemically.

Antibody Titres:

Sera obtained 14 days after the primary immunisations were assayed forIgG and IgM antibody titres to ovalbumin by ELISA assay.

Mouse IgM Titres to Ovalbumin, 14 Days Post Primary Vaccination.

All titres shown are as ELISA optical density at 1/200 serum dilution.

Vaccine formulation DEAE DEAE DEAE Comb. Comb. Comb. dextran dextrandextran Iscomatrix ™ Adj. Adj. Adj. OD at 1/200 0.581 0.895 0.487 0.2750.778 0.571 0.414

Mouse IgG Titres to Ovalbumin, 14 Days Post Primary Vaccination

All titres shown are as ELISA optical density at 1/200 serum dilution.

Vaccine formulation DEAE DEAE DEAE Comb. Comb. Comb. dextran dextrandextran Iscomatrix ™ Adj. Adj. Adj. OD at 1/200 0.753 0.771 0.745 0.4930.495 0.618 0.716

Summary:

The data above shows that immunisation of mice with the soluble antigenOVA in all of the adjuvants tested induced an antibody responses in boththe IgM and IgG isotypes 14 days after a single vaccination.

The combination adjuvant and DEAE-dextran alone formulation elicitedhigher antibody responses than the Iscomatrix adjuvanted vaccine.

To those skilled in the art, it would be expected that a singlevaccination of mice with a soluble antigen in the presence of anadjuvant would give rise predominantly to an IgM response.

The combined adjuvant induced a strong IgG response in mice vaccinatedwith OVA 14 days after a single vaccination. This result supports theprevious published conclusion (Houston et al, 1976) that DEAE-dextranadjuvant shortens the period to switching from an early IgM to IgGresponse and thereby increases the IgG response to vaccination at anearly stage. This also seems to be a property of the combinationadjuvant.

Example 7 Vaccination of Cattle with 2-10 LHRH-DT in CombinationAdjuvant Vaccines and Vaccination Schedule:

Heifers (female cattle) were administered by subcutaneous injection,vaccines formulated with 200 μg 2-10 LHRH conjugate and either:

-   1. DEAE-dextran (200 mg/dose)—50 heifers-   2. Combination adjuvant (150 mg DEAE-dextran with 500 μg Iscomatrix,    Mass ratio 300)—6 heifers.

All vaccines were formulated as a 2 mL dose.

Vaccines were given at week 0 and week 4, with a 2 week post boost bleedbeing taken at week 6.

Site reactions were scored at 2 weeks post boost on a semi-quantitativescale from 0 to 3.

Blood samples were taken by venepuncture of the jugular or tail vein at2 weeks post boost (week 6). Sera were obtained from the blood samplesby allowing them to clot and separation by centrifugation. Serum titresof the IgG isotype were assayed to LHRH by ELISA.

Site Reactions.

Site reaction scores, scored on a scale of:

0 No visible or detectable reaction.1 Reaction only detectable by palpation2 Visible reaction dectable without palpation3 Severe abscessed reaction

2 Weeks Vaccine PB Score Formulation 0 1 2 3 DEAE-dextran 32 7 8 3 50heifers DEAE-dextran + Iscomatrix ™ 1 1 4 0 6 heifers

Summary:

The use of DEAE-dextran as adjuvant resulted in severe site reactions ofscore 3 (abscess) in 3 of the vaccinated cattle.

The combination adjuvant did not induce severe reactions in any of thecattle, with no score 3 lesions occurring.

Antibody Titres:

Serum IgG titres to LHRH at 2 weeks post boost vaccination weredetermined by ELISA.

Vaccine Group Group mean titre Range of titres DEAE-dextran 4181 100-16836 DEAE-dextran + Iscomatrix ™ 15869 1841-51920

Summary:

The combination adjuvant induced high tires in all vaccinated cattlewith a group mean titre of over 15,000. By comparison, the use ofDEAE-dextran alone as adjuvant resulted in a lower group mean titre of4181, with a much lower range of responses.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

-   Aguado, T. et al. Vaccine 17:2321-2328 (1999).-   Awonyi, C. A., Chandrashekar, V., Arthur, R. D., Schanbacher, B. D.    and Falvo, R. E. J. Androl. 9:160-171 (1988).-   Azuma, I. Vaccine 10:1000-1006 (1992).-   Beh, K. and Lascelles, A. K. Immunol. 54:487-495 (1985).-   Bonneau, M., Dufour, R., Chouvet, C., Roulet, C., Meadus, W. and    Squires, E. J. J. Animal Science 72: 14-20 (1994).-   Caraty, A. and Bonneau, M. C. R. Acad. Sci. Paris 303 Series    D:673-683 (1986).-   Cox, J. C. and Coulter, A. R., “Advances in Adjuvant Technology and    Application”, in Animal Parasite Control Utilizing Biotechnology,    Chapter 4, Ed. Yong, W.K., CRC Press (1992).-   Dalsgaard, K., Arch. Gesamte Virusforsch. 44:243.-   Falvo, R. E., Chandrashekar, V. et al. J. Animal Science 63: 986-994    (1986).-   Fraser, H. M. Immunization with Hormones in Reproductive Research:    07-116 (1975).-   Gupta, R. Advanced Drug Delivery Reviews 32:155-172 (1998).-   Hagen, G., Andresen, O., Blichfeldt, T. and Berg, K. A. Proc. 11th    Congress on Animal Production Abstract 493 (1988).-   Hibma M. and Griffin, J. Vet. Immunol. Immunopath. 31:279-287    (1992).-   Hodgkinson, R. M. et al. Aust. J. Biotechnology 4:166-170 (1990).-   Hoskinson, R. M., Rigby, P. E., Mattner, V. L., Huynh, V. L.,    D'Occhio, M. D., Neish, A., Trigg, T. E., Moss, B. A., Lindsey, M.    J., Coleman, G. D. and Schwartzkoff, C. L. Aust. J. Biotechnol.    4:166-170 (1990),-   Houston, W. E. et al. Infect. and Immun. 13:1559-1562 (1976).-   Jeffcoate, I. A., Lucas, J. M. and Crighton, D. B. Theriogenology    18:65-77 (1982).-   Ladd A., Tsong Y. Y., and Thau R. B., American J. Reproductive    Immunology 22: 56-63 (1990).-   Meloen, et al., Vaccine 12: 741-746 (1994).-   Potter, A. A. and Manns, J. G., International Patent Application No.    PCT/CA97/00559 (1997).-   Robertson, I. S., Fraser, H. M., Innes, G. M. and Jones, A. S. Vet.    Record 111:529-531 (1982).-   Sad S., Gupta H., Talwar G. P., and Raghupathy R., Immunology    74:223-227 (1991).-   Wittman et al. Arch. Virol. 47:225-235 (1975).-   Zee, A., Noordegraaf, C. V., Bosch, H., Gielen, J., Bergmans, H.,    Hoekstra, W. and Die, I. Vaccine 13:753-758 (1995).

1. An adjuvant composition consisting of an ionic polysaccharidecomponent and a saponin component, wherein the saponin component isprovided in the form of an immunostimulatory complex.
 2. The adjuvantcomposition of claim 1, wherein an ionic polysaccharide is an ionicdextran.
 3. The adjuvant composition of claim 2, wherein an ionicdextran is DEAE-dextran or QAE-dextran.
 4. The adjuvant composition ofclaim 1, wherein the immunostimulating complex is a protein-freeimmunostimulating complex.
 5. The adjuvant composition of claim 1,wherein the immunostimulating complex comprises cholesterol,phosphatidyl choline, and Quil A.
 6. The adjuvant composition of claim1, wherein the mass ratio of ionic polysaccharide component toimmunostimulating complex component is in the range of about 100 toabout
 140. 7. The adjuvant composition of claim 6, wherein the massratio is about
 125. 8. The adjuvant composition of claim 1, comprisingabout 10 mg of the ionic polysaccharide and about 80 μg of theimmunostimulating complex, wherein the ionic polysaccharide isDEAE-dextran.
 9. The adjuvant composition of claim 1, comprising about100 mg of the ionic polysaccharide and about 800 μg of theimmunostimulating complex, wherein the ionic polysaccharide isDEAE-dextran.
 10. An immunogenic composition comprising an immunogen andthe adjuvant composition of claim
 1. 11. The immunogenic composition ofclaim 10, wherein said immunogen comprises LHRH.
 12. The immunogeniccomposition of claim 11, wherein said immunogen comprises anLHRH-diphtheria toxoid conjugate.
 13. The immunogenic composition ofclaim 12, comprising from about 5 to about 500 μg of LHRH-diphtheriatoxoid conjugate, from about 5 to about 500 mg of the ionicpolysaccharide, and from about 40 to about 4000 μg of theimmunostimulating complex.
 14. A vaccine comprising (i) the immunogeniccomposition of claim 10, and (ii) one or more pharmaceuticallyacceptable carriers.
 15. A method of eliciting an effective immuneresponse in an animal, comprising administering to an animal aneffective amount of the vaccine of claim
 14. 16. The adjuvantcomposition of claim 1, wherein the mass ratio of the ionicpolysaccharide component to the immunostimulating complex component isin the range of 50 to
 300. 17. An adjuvant composition comprising anionic polysaccharide component and a saponin component, wherein thesaponin component is provided in the form of an immunostimulatorycomplex.
 18. The adjuvant composition of claim 17, wherein an ionicdextran is DEAE-dextran or QAE-dextran.
 19. The adjuvant composition ofclaim 17, wherein the immunostimulating complex comprises cholesterol,phosphatidyl choline, and Quil A.
 20. An immunogenic compositioncomprising an immunogen and the adjuvant composition of claim
 17. 21. Avaccine comprising (i) the immunogenic composition of claim 20, and (ii)one or more pharmaceutically acceptable carriers.
 22. A method ofeliciting an effective immune response in an animal, comprisingadministering to an animal an effective amount of the vaccine of claim21.
 23. An adjuvant composition consisting of an ionic polysaccharidecomponent and a saponin component, wherein the saponin component isprovided in the form of an immunostimulatory complex, wherein theimmunostimulatory complex is a cage-like structure.